Method Article
Bacterial expression and protein purification of mini-fluorescence-activating proteins
https://doi.org/10.21203/rs.3.pex-1077/v1
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Bacterial expression and purification of de novo designed mini-fluorescence-activating proteins (mFAPs) are accomplished by genetically fusing mFAP variants to an N-terminal 6xHis tag and optionally to a Tobacco Etch Virus (TEV) protease epitope. The protocol can take less than 24 hours to complete, and allows for downstream in vitro characterization of photophysical properties of mFAPs by equilibration with exogenous fluorogenic compounds.
bacterial expression
protein purification
De novo designed mini-fluorescence-activating proteins1 (mFAPs) are computationally designed β-barrels with eight β-strands totaling ~12.5 kDa without protein tags.
・Plasmid DNA (Novagen, pET29b(+), pET15b) encoding 6xHis-tagged (and optionally TEV epitope-tagged) proteins, transformed into Lemo21(DE3) E. coli (NEB, C2528J)
・Lysogeny broth (LB) (BD Difco, 244610)
・Terrific Broth II (TBII) (MP Biomedicals, 113046031)
・Carbenicillin (Fisher BioReagents, BP2648250)
・Kanamycin (Fisher BioReagents, BP906-5)
・Isopropyl β-D-thiogalactopyranoside (IPTG) (Sigma Aldrich, 367-93-1)
・Phenylmethylsulfonyl fluoride (PMSF) (Sigma Aldrich, 329-98-6)
・Deoxyribonuclease I (DNase I) from bovine pancreas (Sigma Aldrich, 9003-98-9)
・Ni-NTA agarose resin (Qiagen, 30250)
・His MultiTrap HP 96-well plates (GE Healthcare, 28-4009-89)
・QuBit Protein Assay Kit (ThermoFisher Scientific, Q33212)
・6xHis-tagged Tobacco Etch Virus (TEV-6xHis) protease (Supplementary File 1) purified by size-exclusion chromatography in 20.0 mM Tris, 100 mM NaCl, pH 8.00
・Lysis buffer #1 (25.0 mM Tris, 300 mM NaCl, 20.0 mM imidazole, pH 8.00)
・Lysis buffer #2 (20.0 mM Tris, 300 mM NaCl, 30.0 mM imidazole, pH 7.40)
・Lysis buffer #3 (20.0 mM Na2HPO4, 500 mM NaCl, 30.0 mM imidazole, 0.25% CHAPS, 1.00 mM PMSF, 1.00 mg·mL-1 DNase I, pH 8.00)
・Wash buffer #1 (25.0 mM Tris, 300 mM NaCl, 30.0 mM imidazole, pH 8.00)
・Wash buffer #2 (20.0 mM Tris, 500 mM NaCl, 30.0 mM imidazole, 5% glycerol, pH 7.40)
・Wash buffer #3 (20.0 mM Na2HPO4, 500 mM NaCl, 30.0 mM imidazole, 5% glycerol, pH 8.00)
・Elution buffer #1 (20.0 mM Tris, 300 mM NaCl, 500 mM imidazole, 5% glycerol, pH 7.40)
・Elution buffer #2 (25.0 mM Tris, 300 mM NaCl, 250 mM imidazole, pH 8.00)
・Elution buffer #3 (20.0 mM Na2HPO4, 300 mM NaCl, 500 mM imidazole, 5% glycerol, pH 8.00)
・Dulbecco's Phosphate-Buffered Saline without calcium or magnesium (DPBS) (Thermo Scientific, 14190144)
・Phosphate buffered saline (PBS) (25.0 mM phosphate, 150 mM NaCl, pH 7.40)
・Tris buffered saline (TBS) (20.0 mM Tris, 300 mM NaCl, pH 7.40)
・High salt Tev cleavage buffer (25.0 mM Tris, 100 mM NaCl, pH 8.00)
・Low salt Tev cleavage buffer (20.0 mM Tris, 50.0 mM NaCl, pH 7.40)
・250 mL, 2.0 L, and 2.8 L baffled Erlenmeyer flasks (Belloco, 2542-00250)
・Motorized serological pipette controller (ThermoFisher, 14-387-163)
・Micropipettes (Rainin Instruments, 17014382, 17014384, 17014388, 17014393)
・Serological pipette tips (ThermoFisher, 13-676-10H, 13-678-11E, 13-676-10K, 13-676-10R)
・Micropipette tips (VWR, 89136-576, 76175-406, 76175-412)
・Orbital platform shaker in 37°C incubator (Eppendorf, M1324-0000)
・Vibrating platform shaker in 37°C incubator (Fisher Scientific, 13-889-420)
・Floor centrifuge (Beckman Coulter, Avanti JXN-26) with fixed-angle rotor (Beckman Coulter, J-LITE JLA-8.1000) and 1 L bottles (Beckman Coulter, 363688)
・Countertop centrifuge (Thermo, Sorvall Legend XTR), with plate rotor (Thermo Scientific, 750003624) and fixed angle rotor (Thermo Scientific, 75003663)
・Sonicator with probe (Qsonica, Q500, 562-A, 525-A)
・Microplate sonicator (Qsonica, Q500, CL-334)
・Microfluidizer (Microfluidics, M110P)
・FastPrep-96 (MP Biomedicals)
・Glass beads, unwashed (Millipore Sigma, G9143), for FastPrep-96
・Immobilized metal affinity chromatography (IMAC) columns (Bio-Rad, 7321010)
・Falcon conical centrifuge tubes, 50 mL (Corning, 352070), for cell lysates and IMAC sample collection
・Amicon Ultra-15 Centrifugal Filter Units with 3 kDa cutoff (Millipore Sigma, UFC900324)
・Nunc 2.0 mL DeepWell 96-well plates, sterile (Thermo Scientific, 278743)
・Skirted 96-well polymerase chain reaction (PCR) plates (ThermoScientific, AB2396)
・Adhesive Breathe-Easy film covers (USA Scientific, 9123-6100), for Nunc 2.0 mL DeepWell 96-well plates
・Adhesive foil covers (ThermoScientific, AB-0626), for skirted 96-well PCR plates
・High-performance liquid chromatography (HPLC) machine (Agilent Technologies, LC 1200 Series)
・Superdex 75 Increase 10/300 GL column (Cytiva, 29148721)
・Superdex 200 Increase 10/300 GL column (Cytiva, 28990944)
・QuBit 2.0 fluorometer (ThermoFisher Scientific, Q32866)
・NanoDrop 8000 spectrophotometer (Thermo Scientific, ND-8000-GL)
Large-scale bacterial expression and protein purification.
1. For expression of 6xHis-tagged proteins encoded on pET15b or pET29b(+) vectors in Lemo21(DE3) E. coli, either a single colony from an agar plate, 25.0 μL of overnight-grown culture, or 25.0 μL of a 20% glycerol culture stock is inoculated into 50.0 mL, 500 mL, or 1.00 L of lysogeny broth (LB) media or Terrific Broth II (TBII) media supplemented with 50.0 μg·mL-1 carbenicillin (pET15b) or 50.0-100.0 μg·mL-1 kanamycin (pET29b(+)) at final concentration.
2. To induce protein expression, cells are grown at 37°C shaking at 200-250 rotations per minute (rpm) overnight (16-22 hours) with 500 µM isopropyl β-D-thiogalactopyranoside (IPTG) final concentration added either prior to inoculation or 4 hours prior to harvesting cells.
3. Cells are harvested by centrifugation at 3,000-4,000xg for 10 minutes at 4°C.
4. Cell pellets are resuspended in 20.0-30.0 mL of either lysis buffer #1 supplemented with 1.00 mg·mL-1 phenylmethylsulfonyl fluoride (PMSF) and a small amount of deoxyribonuclease I (DNase I), lysis buffer #2, or lysis buffer #3.
5. Cells are lysed via either sonication at 40-70% amplitude, FastPrep-96 at 1,600 rpm with glass beads, or cell disruption using a microfluidizer at 18,000 psi.
6. Crude cell lysates are centrifuged at 24,000xg for 30-60 minutes at 4˚C.
7. To purify clarified cell lysates via gravity Ni-NTA immobilized metal affinity chromatography (IMAC), 2.00 mL of Ni-NTA agarose resin is pre-equilibrated with 10.0 mL of either lysis buffer #1, wash buffer #2, or wash buffer #3.
8. Clarified cell lysate is loaded onto the column and the flow-through is collected.
9. The column is washed 1-3 times with 20.0 mL of either wash buffer #1, wash buffer #2, or wash buffer #3.
10. The proteins are eluted from the column in 5.00-15.0 mL of either elution buffer #1, elution buffer #2, or elution buffer #3 and the eluates are collected.
11a. For keeping 6xHis tags intact, IMAC-purified proteins are buffer exchanged into high salt Tev cleavage buffer or low salt Tev cleavage buffer using Amicon Ultra-15 Centrifugal Filter Units. Proceed to step 14.
11b. For removing 6xHis tags, IMAC-purified proteins are concentrated to ~1.0 mL using Amicon Ultra-15 Centrifugal Filter Units, 1.0-5.0 mg·mL-1 6xHis-tagged Tobacco Etch Virus (TEV-6xHis) protease is added at a ~1:100 dilution, and the reaction is incubated at 25°C overnight. Proceed to step 12.
12. TEV-6xHis protease is removed via secondary Ni-NTA IMAC purification, eluting proteins in 5.00 mL of lysis buffer #1 or lysis buffer #2.
13. Cleaved protein is concentrated in Amicon Ultra-15 Centrifugal Filter Units.
14. Protein concentrations are measured using either a QuBit 2.0 and QuBit Protein Assay Kit or a NanoDrop 8000 with extinction coefficients predicted from amino acid sequence using the ProtParam tool2 or Biopython module3.
Size-exclusion chromatography (SEC).
1. IMAC-purified proteins are prepared at ≥1 mg·mL-1 and applied to a Superdex 75 Increase 10/300 GL column or Superdex 200 Increase 10/300 GL column on a LC 1200 Series high-performance liquid chromatography (HPLC) machine for separation of IMAC-purified proteins based on molecular size.
2. SEC-purified proteins are eluted in either high salt Tev cleavage buffer, Tris buffered saline (TBS), phosphate buffered saline (PBS), or Dulbecco's Phosphate-Buffered Saline without calcium or magnesium (DPBS) (Thermo Scientific).
3. Monomeric protein fractions are collected and concentrated using Amicon Ultra-15 Centrifugal Filter Units with 3 kDa cutoff.
4. Protein concentrations are measured as in step 14 of "Large-scale bacterial expression and protein purification."
Small-scale bacterial expression and protein purification.
1. 25.0 μL of overnight-grown cultures of Lemo21(DE3) E. coli are inoculated into each well of Nunc 2.0 mL DeepWell 96-well plates containing 1.00 mL of LB media supplemented with 50.0 μg·mL-1 carbenicillin (pET15b) final concentration, and plates are sealed with adhesive Breathe-easy film covers. Optionally, 2-8 replicate plates are prepared to optimize purified protein yields.
2. Cells are grown at 37°C and shaken at 1,200 rpm for 3-4 hours.
3. 500 µM IPTG final concentration is added to each well.
4. Protein expression is induced for 4 hours at 37°C shaken at 1,200 rpm.
5. Cells are pelleted at 2,272xg for 2-5 minutes in a plate centrifuge.
6. Pellets are resuspended in 50.0 μL of lysis buffer #1 supplemented with 1.00 mg·mL-1 PMSF and a small amount of DNase I, combining pellets of any replicate plates.
7. Cells are transferred to skirted 96-well polymerase chain reaction (PCR) plates and sealed with adhesive foil covers for cell lysis via a microplate sonicator at 40-60% amplitude for 2-4 minutes in an ice bath.
8. Crude cell lysates are centrifuged at 2,272xg for 5 minutes in a plate centrifuge.
9. Clarified cell lysates are applied to His MultiTrap HP 96-well plates pre-equilibrated in lysis buffer #1, and IMAC purification undergone following the manufacturer's protocol for purification except resin is washed three times in wash buffer #1 and eluted three times, each time using 50.0 μL of elution buffer #1.
10. Eluates are consolidated into a single 96-well PCR plate.
Large-scale bacterial expression and protein purification – 18-48 hours
Size-exclusion chromatography (SEC) – 30-60 minutes
Small-scale bacterial expression and protein purification – 8-10 hours
IMAC-purified and SEC-purified protein samples for downstream in vitro characterization.
1. Dou, J., Vorobieva, A.A., Sheffler, W. et al. De novo design of a fluorescence-activating β-barrel. Nature 561, 485–491 (2018). https://doi.org/10.1038/s41586-018-0509-0
2. Gasteiger, E. et al. Protein Identification and Analysis Tools on the ExPASy Server. The Proteomics Protocols Handbook 571–607 (2005). https://doi.org/10.1385/1-59259-890-0:571
3. Cock, P. J. A. et al. Biopython: freely available Python tools for computational molecular biology and bioinformatics. Bioinformatics 25, 1422–1423 (2009). https://doi.org/10.1093/bioinformatics/btp163
J.C.K. would like to thank U. Nattermann, A. Courbet, K.-L. Herpoldt, S. Boyken and other members of the Institute for Protein Design for helpful discussions. This material is based upon work supported by the National Science Foundation Graduate Research Fellowship Program under Grant No. DGE-1256082. Any opinions, findings, and conclusions or recommendations expressed in this material are those of the authors and do not necessarily reflect the views of the National Science Foundation. The authors declare the following competing interests: J.C.K., J.D., A.A.V. and D.B. are coinventors on patent application PCT/US2019/025743. J.C.K. and D.B. have filed a provisional patent application 63/116,875.
Supplementary File 1. Tobacco Etch Virus (TEV) protease amino acid sequence used in this protocol in FASTA format.
This protocol has been posted on Protocol Exchange, an open repository of community-contributed protocols sponsored by Nature Portfolio. These protocols are posted directly on the Protocol Exchange by authors and are made freely available to the scientific community for use and comment.
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